The Harbinger
August 22, 2005
Hiram Undergrads Present Research Findings in Indiana
Nine
student researchers (pictured), along with Dr. Brad Goodner, presented
their experimental findings in one oral and six poster presentations at
the 26th Annual Crown Gall Conference. This meeting brings
researchers from all over the U.S. and around the world to discuss the
biology of the plant pathogen and biotechnology agent Agrobacterium
tumefaciens. The titles and abstracts of the presentations are on
the attached pages. One of the presentations, by Terrence Johnson
and DaJuan Whiteside, was awarded the Midwest Scientific Award for best
undergraduate poster.Summaries of the presentations are below:
Evolutionary Insights from the Agrobacterium Genome Project
Brad Goodner, students in 2005 Genetics course, & the K84/S4 Genome Consortium
Department of Biology, Hiram College
The K84/S4 Genome Consortium has tripled the genomic information available for the genus Agrobacterium. In-depth analysis of each genome along with comparison to the many other sequenced genomes within the Rhizobiaceae and the larger alpha-Proteobacteria will provide further insight into how Agrobacterium strains interact with plants and survive in a wide variety of soil conditions. However, the same comparative analysis also yields much information on genome evolution within the class, family, and genus. I will focus on three aspects of genome evolution: 1) apparent lateral gene transfer events that predate the radiation of the alpha-Proteobacteria, 2) genomic rearrangements within the genus Agrobacterium, and 3) relatively recent events within the biovar 1 clade (includes the type strain C58). At several points, I will make connections to ongoing projects at Hiram College.
Further Links Between Sugar Metabolism & Osmotic Tolerance in Agrobacterium
Mandy Reed, Virginia Mateo, Ben Shelton, Jessica Edwards, Frank Arnold, Joshua Collins, Cathy Wheeler, & Brad Goodner, Department of Biology, Hiram College
Previous work in our lab has shown that while there is functional redundancy for sucrose catabolism in A. tumefaciens C58, the gene annotated as sucrose hydrolase (AGR_C_1721/Atu0944) is important for osmotic tolerance. Our current hypothesis is that sucrose hydrolase, unique to biovar 1 strains, actually catalyzes the synthesis of mannosucrose which is known to be accumulated under osmotic stress. We will present the current status of our efforts to test this hypothesis.
To further explore the genetic basis for osmotic tolerance, we conducted a large scale screen for mutants of A. tumefaciens C58 (biovar 1) and A. vitis S4 (biovar 3) unable to grow either under high salt conditions or with low levels of sucrose as a sole carbon source. As we will show in this poster, most of the mutants characterized so far have substantiated the link between sugar metabolism and osmotic tolerance.
Finally, we will update our efforts to characterize the basis of the 3-ketosucrose pathway in A. tumefaciens C58.
Functional Differences Between the Two Aconitases of A. tumefaciens C58
Terrence Johnson, DaJuan Whiteside, Leora Livingston, Razan Yasin, Gena Dottle, & Brad Goodner, Department of Biology, Hiram College
Aconitases are monomeric iron-sulfur proteins whose primary function is to catalyze the interconversion of citrate to isocitrate in the citric acid cycle. Most members of the Bacteria domain have two aconitases, AcnA and AcnB. According to a model developed in E. coli and B. subtilis, a second function of aconitases is to monitor oxidative stress and post-transcriptionally regulate the synthesis of additional aconitase protein as well as cellular processes such as motility. In E. coli, AcnA is induced during periods of stress while AcnB is the major enzyme involved in exponential growth. Mutations in acnA have no impact on motility, while mutations in acnB decrease motility.
Unlike the E. coli model, the sequenced members of the alpha-Proteobacteria have only a acnA gene except for A. tumefaciens C58 which has both acnA and acnB. To analyze the role of the two aconitases in A. tumefaciens C58, we generated mutations in each gene (AGR_C_4866 = acnA; AGR_L_294 = acnB). Contrary to the E. coli model, the acnA- mutant is an auxotroph on minimal media and it shows a very hypermotile phenotype on rich medium. The acnB- mutant shows only a mild hypomotile phenotype. The hypermotility of the acnA- mutant does not substantially impact virulence, due to the repression of motility by low pH conditions. We will present data on growth, motility, and virulence, as well as other experiments to dissect the role of the two aconitases.
Unearthing the Linearization of Chromosome II in the Agrobacterium Biovar 1 Clade
Erin Henry, Allison Sabo, Erin Telepak, & Brad Goodner, Department of Biology, Hiram College
Past genetic/physical mapping has shown that Agrobacterium biovar 1 and biovar 3 strains harbor two chromosomes, and the complete sequence of A. tumefaciens C58 provided strong evidence for the plasmid origin of chromosome II. However, the chromosome II of biovar 1 differs dramatically in its topology (linear with hairpin loop ends) from that of its homolog in biovar 3 (circular). We have found that all biovar 1 strains tested so far and some closely related Rhizobium strains have both a linear chromosome II and the gene now known to encode the enzyme required for hairpin loop maintenance.
The simplest hypothesis to explain the origin of the linearity of chromosome II in the biovar 1 clade and a system for its maintenance is one where both the hairpin loops and the protelomerase gene entered the genome at the same time. Our working model proposes that an illegitimate recombination occurred between the circular form of chromosome II and a linear phage genome. No such truly linear Agrobacterium-specific phage has ever described in the literature, only lambdoid-type phages with cohesive ends that form a circle in vivo. We will discuss two strategies we are using to look for Agrobacterium-specific phages, with the hopes of finding phages with linear genomes. Our first strategy is a traditional phage search of plant tumor and soil samples using several Agrobacterium strains as hosts. We have not had a tremendous amount of success and welcome any suggestions for how to improve this strategy. Our second strategy is a PCR-based approach that should identify any biovar 1 strain or phage in an environmental sample that contains a protelomerase gene. After isolation of total DNA from samples, step one is a straightforward PCR using primers to amplify an internal portion of the protelomerase gene. For any samples with success in step one, total DNA is cut and circularized, then used for inverse PCR with primers reading out from near each end of the protelomerase gene. Successful amplification will yield the sequences surrounding the protelomerase gene. Knowledge of the gene neighborhood will help us dissect the evolutionary history of the protelomerase gene and may eventually yield a phage-like neighborhood consistent with our working model.
Initial Characterization of Nitrogen Metabolism in A. tumefaciens C58
Nabil Abraham, Ian Bennett, Telisha Law, Frank Arnold, Josh Collins, Cathy Wheeler, & Brad Goodner, Department of Biology, Hiram College
Nitrogen metabolism in Agrobacterium is quite complex. The organism has a wide array of mechanisms for using inorganic and organic forms of nitrogen for its basic anabolic needs as well as for anaerobic respiration (using nitrate as electron acceptor). We are currently focusing on the roles of the two different nitrate reductases in the A. tumefaciens C58 genome. Using gene disruption mutations in the genes encoding the large subunits of the NAS-type and NAP-type nitrate reductases, we have found and will present some evidence for these enzymes having overlapping functions.
A much more controversial question is whether Agrobacterium can grow in the absence of any added nitrogen in the growth medium. This question of nitrogen fixation/nitrogen scavenging (we are unable to separate the two possible phenomena at this point) came to our doorstep in the form of Agrobacterium strain UK1. This strain was pulled out of a stream bank in the United Kingdom as a potential free-living N-fixer. While its initial phenotype suggested it was Azotobacter, its 16S rRNA sequence said otherwise and we have shown that it is clearly a biovar 1 Agrobacterium. This strain and the sequenced A. tumefaciens C58 can grow in minimal medium minus nitrogen in aerobic and microaerophilic conditions. We will present evidence that this is real growth not due to nutrient carryover. Since the C58 genome clearly shows the absence of known nitrogenase subunits, the observed fixation/scavenging must be due to some novel biochemical reactions. We are currently using a mutant hunt approach to identify the genes required for these reactions and will present the current status of our search.
Update on Agrobacterium Functional Genomics Studies at Hiram College
Frank Arnold, Divya Balasubramanian, Josh Collins, Dan Factor, Stephanie Larrick, Chris Penton, Becky Roemer, Andee Wilson, Lindsey Wilson, Sarah Zilka, students in 2002-2005 Molecular and Cellular Biology courses, Prudy Hall, Cathy Wheeler, & Brad Goodner, Department of Biology, Hiram College
Obtaining the complete genome sequence of any organism is rather like finding a hidden library from another civilization - you know there is important information inside but cannot make sense of all it right away. The genome sequence of A. tumefaciens C58 yielded many genes we completely expected, some genes that we are not too surprised to see, some surprising finds, and some enigmas/wild goose chases (only time and experiments will tell). Starting in the spring of 2002, both within courses and through independent projects, we have been experimentally testing the putative functions of several genes each year. The putative functions of most of the genes tested arose from the published sequence annotation, however a few of them came out of forward genetic mutant hunts, and one came out of the lack of bioinformatic support for an assayable function. In this poster, we will present short updates on a wide variety of genes involved in central metabolism, metabolism of amino acids, sugars, and polysaccharides, and stress responses.
Update on Genome Projects Involving Hiram College
Divya Balasubramanian, Adam Ewing, Dan Factor, Stephanie Larrick, Becky Roemer, Andee Wilson, Lindsey Wilson, students in 2004-2005 Biochemistry & Genetics & Molecular and Cellular Biology courses, Prudy Hall, Cathy Wheeler, & Brad Goodner, Department of Biology, Hiram College
Since the publication of the A. tumefaciens C58 genome in late 2001, we have been privileged to participate in several other genome projects. Our contributions vary with each project depending on the needs of the project and how it fits in with courses at Hiram College, and range from the generation of physical and genetic maps of chromosomes and plasmids to gap closure to sequence annotation.
Two more strains of Agrobacterium, the grape pathogen A. vitis S4 and the well known biocontrol agent A. radiobacter K84 are being sequenced by the K84/S4 Genome Consortium and are now in the annotation phase. We confirmed the number and sizes of the genomic components of strain S4, used bioinformatics-based approaches to help with the assembly of both genomes, and are currently assisting in the annotation of both genomes.
Two species of Xenorhabdus, X. bovienii and X. nematophila, are being sequenced by the Xenorhabdus Genome Consortium. These bacteria pull off a Jeckyl and Hyde - they form mutualistic associations with certain nematodes and then together kill particular insect larvae to use as nutrient sources for rapid proliferation. The X. bovienii genome is in the annotation phase and X. nematophila is very close to closure of the genome sequence. Our main job here is annotation, although we have also helped with the assembly of both genomes.
Chromohalobacter salixegens (formerly Halomonas elongata) is one of the most halotolerant members of the domain Bacteria. The DOE Joint Genome Institute provided draft sequence of its genome and we are now collaborating with Dr. Laszlo Csonka at Purdue University and the DOE-JGI to finish the genome and annotate it. We are providing a partial physical/genetic map of the genome, bioinformatics-based gap closure, and sequence annotation.
Azotobacter vinelandii is a well known, free-living, aerobic nitrogen fixer. A draft of its genome has been done by DOE-JGI and we are now part of a recently funded Azotobacter Genome Consortium to finish and annotate the genome. In preparation for those efforts, we are currently starting a physical/genetic map of the genome.
Homrighouse serves Hiram for over a decade
Randy Homrighouse has resigned after 11 1/2 years of service as Director of Human Resources. "I never thought I would be here this long," Homrighouse said. "This is a beautiful campus, but it's the people that have kept me here. I have really enjoyed my association with all aspects of the people part of human resources - the faculty, staff, retirees, trustees and alumni. Everyone is unique and special and has something to bring to the community."
Homrighouse has worked for 31 years in human resources, but only with two employers. "I had never worked in higher education before coming to Hiram. I really appreciated how great everyone was to me as I learned the nuances of higher education. I feel that together we have accomplished a lot in terms of providing employees with an office that has their best interests in mind and one where they can go to for confidential and professional services."
Continued Homrighouse, "I'm looking forward to having more time to spend in areas of my life that interest me the most. I've always enjoyed Northeast Ohio, so I plan to stay in the area. I'm a buckeye! I will miss the day to day interactions I have enjoyed with everyone here. But I'll be close enough to celebrate the successes that will be occuring here because of the good work we've done together."
Hiram Orchestra Resumes Practice
The Hiram Chamber Orchestra will hold its first rehearsal on Thursday evening, September 1, 2005 at 7:00 in Frohring Recital Hall on the Hiram College campus. Anyone with experience playing violin, viola, cello or bass is welcome to join the group. Last year, the Hiram Chamber Orchestra consisted of 16 players and has the potential for even more this year. Music is selected to gently challenge the group and provide an enjoyable musical experience for everyone. Last year, works by Ravel, Pachelbel, Bartók, and Purcell were performed. For more information about the Hiram Chamber Orchestra, contact Randall Fusco at 330.569.5298, or by e-mail at fuscorj@hiram.edu.
The Pelican wraps up the Tyst summer theater series
In
its latest production, Tyst is excited to produce one of August
Strinberg’s final and most evocative chamber plays. The Pelican will
run through August 28 (this weekend) in Hiram’s Bates Hall. Performances will be
Thursdays, Fridays, and Saturdays at 8 p.m. and Sundays at 2 p.m.In this intriguing and ominous work, The Pelican introduces a family of sleepwalkers, a family whose very existence has been built on deception. As each family member begins to wake up, the myth of motherly sacrifice is exposed and revenge is dealt out with ghastly consequences.
The cast includes actors from throughout Northeast Ohio. Hiram Professor of English Ellen Summers plays the cunning matriarch of the family. Justin Tatum, a Shaker Heights resident, plays her neglected and sickly son, Frederick. Case Western Reserve University student Kristie Erin Barnes plays her naïve and temperamental daughter, Gerda; and Gerda’s unscrupulous husband, Axel, is played by Ohio City resident Tom Kondilas. The cast is rounded out by Akron resident Sue Antel who plays the brazen housemaid, Margaret. The production is directed by Cleveland resident Adrienne Moon.
Strindberg is Sweden’s master playwright, novelist, and short-story writer. Combining psychology, naturalism and mysticism, his plays drew on the problems of his marriage and his interest in self-analysis. Moon is the lead director at Dobama’s Night Kitchen in Cleveland Heights.
Hiram College faculty, staff, and students can receive four free tickets to any performance of The Pelican by calling x 5900.